Recent biotechnological advances have facilitated identification and isolation of components in complex antigens which provide prospects for successful development of safe and practical vaccines. Often, however, these isolated components are not as immunogenic as the complete complex antigens from which they were derived. In order to enhance an immune response to a weakly antigenic immunogen in a recipient animal, adjuvants are frequently administered with the immunogen. Despite the universal acceptance of adjuvants, however, the number suitable for use in humans is limited.
Ideally, an adjuvant should potentiate long-lasting expression of functionally active antibodies, elicit cell-mediated immunity (CMI), and enhance production of memory T- and B-lymphocytes with highly specific immunoreactivity against an invading antigen. In addition to providing a defense upon immediate challenge with an foreign antigen, these responses should provide protection against any future encounters of the host with a specific antigen. More important is the ability of an adjuvant to augment the immune response with a minimum of toxic side effects. Therefore, efficacy of an adjuvant is described in terms of how it balances positive (potentiated immunity) and negative (toxicity) influences.
Controlled immunization for the purpose of stimulating antibody production by B cells is dependent upon a myriad of factors inherent to both the antigen itself and the immunized animal. In general, the farther removed in evolutionary terms the antigen, or its source, is from the invaded host, the more effective the immune response elicited by the antigen. Antigens derived from closely related species are less competent in eliciting antibody production due to the fact that the host immune system is sometimes unable to clearly distinguish the foreign antigen from endogenous, or self antigens. In addition, the dosage of the antigen, the purity of the antigen, and the frequency with which the antigen is administered are also factors which significantly contribute to the resulting antibody titer and specificity of the resulting antibodies. Still other factors include the form, or complexity, of the antigen, and how the antigen is administered. Finally, both the genetic makeup and overall physiological state of the immunized animal contribute to the extent to which an immune response is mounted. Of these factors, the form or complexity of the antigen is directly affected by immunization with an adjuvant.
Current understanding suggests that adjuvants act to augment the immune response by a variety of different mechanisms. In one mechanism, the adjuvant directly stimulates one of either CD4.sup.+ helper T-cell subpopulations designated T.sub.H 1 or T.sub.H 2 [Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173 (1989)]. Helper T cells are required for B-cell antibody responses to most antigens. In an appropriate immune response, an antigen is captured and processed by an antigen-presenting cell (APC), e.g., circulating or tissue macrophages, and presented on the surface of the APC in association with a class II major histocompatibility (MHC) molecule. In this form, the antigen can interact with receptors on the surface of helper T cells thereby activating the particular subpopulation of cells to express and secrete any of a number of cytokines. The nature of cytokine production depends on the subset of helper T cells activated, a result that can be modulated in part by the choice of adjuvant. For example, alum, an aluminum salt adjuvant approved for clinical use in humans, has been reported to selectively activate T.sub.H 2 cells in mice [Grun and Maurer, Cell. Immunol. 121:134-145 (1989)], while Freund's complete adjuvant (CFA), an emulsion of mineral oil with killed mycobacteria [Freund, et al., Proc. Soc. Exp. Biol. Med. 37:509 (1937)], preferentially activates murine T.sub.H 1 cells [Grun and Maurer, Cell. Immunol. 121:134-145 (1989)].
Another mechanism by which the immune response is augmented involves the direct stimulation of B cells by, for example, lipopolysaccharide (LPS) from Gram-negative bacteria. [Gery, et al., J. Immunol. 108:1088 (1972)]. LPS has also been shown to stimulate secretion of interferon-.gamma. (INF-.gamma.) [Tomai and Johnson, J. Biol. Resp. Med. 8:625-643 (1989)], which both inhibits proliferation of T.sub.H 2 cells and stimulates differentiation of T.sub.H 1 cells [Gajewski, et al., J. Immunol. 143:15-22 (1989); Gajewski, et al., J. Immunol. 146:1750-1758 (1991)]. The mechanism by which LPS potentiates the immune response is therefore through direct stimulation of B cells, and indirect regulation of both T.sub.H 1 and T.sub.H 2 cell populations.
Still other modes of immunopotentiation have been reported for other adjuvants. Oil emulsions (i.e., Complete Freund's Adjuvant [CFA], Freund's incomplete adjuvant [FIA]) and liposomes act through depot formation as does alum, thus allowing for slow release of antigen. Slow release of antigen permits extended exposure of the antigen to the immune system and also allows for initial immunization with a dosage of antigen that, if delivered at one time, would ordinarily be counterproductive to antibody formation. It has been previously reported that while a large initial dose of antigen results in the production of a higher immediate titer of antibody, the increase in antibody titer and increase in antibody specificity as a function of time is not as great as observed with lower and more frequent doses of antigen [Siskind, G., Pharm. Rev. 25:319-324 (1973)]. Therefore, adjuvants which control presentation of an antigen to the immune system modulate antigen dosage in addition to altering the form, or complexity, of the antigen.
To date, only one adjuvant, alum [AlK(SO.sub.4).sub.2.H.sub.2 O], has proven sufficiently non-toxic to permit its use in humans. Alum not only acts through T.sub.H 2 cell activation, depot formation and slow release of antigen following immunization [Edelman, Rev. Infect. Dis. 2:370-383 (1980); Warren, et al., Ann. Rev. Immunol. 4:369-388 (1986)], but also through granuloma formation by attracting immunocompetent cells [White, et al., J. Exp. Med. 102:73-82 (1955)] and activation of complement [Ramanathan, et al., Immunol. 37:881-888 (1979)]. However, alum is not without its negative side effects which include erythema, subcutaneous nodules, contact hypersensitivity, and granulomatous inflammation. Other adjuvants, which are widely employed outside of human application, are also the focus of continuing research to develop acceptable alternatives for use in humans. Included are the above mentioned oil emulsions (i.e., CFA and FIA), bacterial products (i.e., LPS, cholera toxin, mycobacterial components and whole killed Corynebacterium parvum, Corynebacterium granulosum, and Bordetella pertusis, liposomes, immunostimulating complexes (ISCOMs), and naturally occurring and derivatized polysaccharides from other than bacterial sources.
The immunopotentiating capacity of polysaccharides has been a focus of investigation over the past few years as these compounds are widespread in nature, e.g., as structural components in the cell walls of bacteria, and exoskeletons of insects and crustacea. Lipopolysaccharide (LPS) isolated from certain Gram-negative bacteria is one such polysaccharide even though the adjuvant properties of LPS are derived mainly from the lipid A region of the molecule, and not from the o-specific polysaccharide or core oligosaccharide regions of the molecule. LPS, which augments both humoral [Johnson, et al., J. Exp. Med. 103:225-246 (1956)] and cell-mediated immunity [Ohta, et al., Immunobiology 53:827 (1984)], possesses numerous biological activities, but is impractical for use in humans due to its inherent toxicity as reviewed by Gupta, et al., Vaccine 11:291-306 (1993). Attention has therefore shifted to other polysaccharides including, among others, chitosan.
Chitosan [.beta.-(1-4)-2-amino-2-deoxy-D-glucan] is a derivative of chitin and has been widely used in biomedical applications, due in part to is biodegradability by lysozyme and low toxicity in humans. These same properties have resulted in increased interest in chitosan as an immunopotentiating agent. For example, Matuhashi, et al., in U.S. Pat. No. 4,372,883, disclosed conjugation of soluble polysaccharides, including chitosan, to normally toxic antigens, conjugation thereby detoxifying the antigen and permitting its use as an immunogen. Matuhashi et al., however, did not address the use of insoluble forms of chitosan, nor did Matuhashi compare the resulting serum antibody titer with that obtained from immunization with other known adjuvants.
Likewise, Suzuki, et al., in U.S. Pat. No. 4,971,956, disclosed the use of water soluble chitosan-oligomers as therapeutics for treatment of bacterial and fungal infections, as well as for the treatment of tumors. Suzuki, et al, discussed the difficulty in modifying chitosan to produce an appropriate water soluble form, disclosing that water-insoluble forms are impractical for therapeutic application. In addition, Suzuki et al., does not disclose conjugation of an antigen to chitosan to effect enhanced immune response.
Mitsuhashi, et al., in U.S. Pat. No. 4,814,169, disclosed the use of human protein conjugated to soluble polysaccharides, including chitosan, to generate antibodies against human protein in non-human animals. Administration of the human protein/polysaccharide solution was by intravenous, intraperitoneal, or subcutaneous injection. Other routes, including oral and rectal administration, were not addressed in the disclosure.
Nishimura, et al. [Vaccine 2:93-99 (1984)] reported the immunological properties of derivatives of chitin in terms of activation of peritoneal macrophages in vivo, suppression of tumor growth in mice, and protection against bacterial infection. Results suggested that both chitin and chitosan were ineffective stimulators of host resistance against challenge with tumor cells or bacteria, but that chitosan moderately induced cytotoxic macrophages. Results with modified, de-acetylated chitosan, which forms a gel in an aqueous environment, was shown to more effectively activate macrophages, suppress tumor growth and stimulate resistance to bacterial infection.
Marcinkiewicz, et al., [Arch. Immunol. Ther. Exp. 39:127-132 (1991)] examined the immunoadjuvant activity of water-insoluble chitosan and reported significant enhancement of T-dependent humoral response, but only moderate augmentation of T-independent humoral response. The enhanced humoral response was detected with chitosan at doses of 100 mg/kg administered either intravenously or intraperitoneally. Subcutaneous and oral administration were specifically reported as being ineffective. In addition, Marcinkiewicz, et al., does not suggest conjugation of an antigen to insoluble chitosan, stating that chitosan "resulted in the same response irrespective of the site of administration--either together or separately from antigen."
In light of the fact that only one existing adjuvant has been approved for use in humans, there thus exists a need in the art to provide novel and less toxic adjuvants for potential application in humans. Improved adjuvants will permit the production of more effective vaccines and will improve the production of monoclonal antibodies with therapeutic potential.